Smallpox, also called variola, is one of infectious viral diseases which were once most feared. Viruses classified into Orthopoxvirus genus including variola virus have a genome of about 180 to 200 kbp in length, which has a region of about 100 kbp in its central part and well preserved among viruses classified into Orthopoxvirus genus. Since protective antigens are coded in this region, viruses classified into Orthopoxvirus genus exhibit almost complete cross immunity (see e.g. Non-patent reference 1) among them. Generally, an attenuated strain derived from a pathogenic virus is used for the manufacturing of a live vaccine. In case of smallpox vaccines (variola vaccines), however, vaccinia viruses, not the viruses derived from smallpox viruses, typically, Lister strain, New York City Board of Health strain (hereinafter also referred to as “NYCBH strain”), Dairen strain, Ikeda strain, etc. have been used.
In Japan, smallpox vaccines, which were manufactured from bovine skin tissues with vesicles generated by inoculating the Lister strain or the Dairen/Ikeda strain to cattle, had been used. These vaccines had caused serious side effects such as postvaccinal encephalitis in infants receiving the first dose at a ratio of several to a million (see e.g. Non-patent reference 2). They were not sterile, as cattle were used for the propagation and passage, and contained non-purified viruses. Thus, Hashizume et al. prepared temperature-sensitive attenuated virus strains originated from the Lister strain adapting the virus to primary cells of rabbit kidney and to a lower temperature. In the end, the LC16m8 strain, which formed small pocks in the chorioallantoic membrane of chicken egg, was selected from clones and purified. The LC16m8 strain was approved in Japan in 1975 after clinical trials (see e.g. Non-patent reference 3). Although the attenuated vaccinia virus LC16m8 strain has a extremely lower neurovirulence than that of its parent strain, the Lister strain, or the NYCBH strain which is a parent strain of a smallpox vaccine Dryvax approved in the U.S. (see e.g. Non-patent references 3 and 4), the level of neutralizing antibodies induced by the LC16m8 strain was almost comparable to the level of those induced by the Lister strain (see e.g. Non-patent reference 5). Also, it is only the LC16m8 strain that is approved as an attenuated smallpox vaccine manufactured in cell culture.
The LC16m8 strain was selected as a virus which formed small pocks after being passaged for three times at a lower temperature originated from its parent strain, the LC16mO strain, and was cloned. It has been revealed that the cause of forming such small pocks is the deficiency of B5R protein due to a frame-shift mutation occurred in the B5R gene (see e.g. Non-patent reference 6). It is reported that, since the B5R protein is a major constituent protein of the extracellular membrane of the viruses classified into Orthopoxvirus genus, the deficiency of this protein may results in the reduced efficiency of viral infection to neighboring cells and thereby in the small pocks (see e.g. Non-patent reference 7).
On the other hand, since the deficiency of B5R protein is due to the deletion of a single nucleotide in the B5R gene, LC16mO-typed reversionary strains (revertants) appear comparatively shortly (see e.g. Non-patent reference 8). A nucleotide sequence analysis of the B5R gene of these revertant strains revealed that the reading frame of ORF of B5R was reverted due to a duplication of a single nucleotide, a duplication of four nucleotides or a deletion of two nucleotides in the adjacent of the nucleotide 274G. When compared on biological properties, the revertant strains formed plaques that were smaller in size than those of the LC16mO strain but larger than those of the LC16m8 strain in RK-13 cells and Vero E6 cells. They also formed pocks on the chorioallantoic membrane of embryonated chicken egg equivalent in size to those of LC16mO. There was no significant difference in the proliferative ability of the virus in rabbit skin, which is thought to be correlated to the proliferative ability in human skin, when the revertant content was up to around 4%. Meanwhile, it was presumed that there was a significant difference in the proliferative ability when the revertant content became much higher (see e.g. Non-patent reference 9).
Non-patent reference 1: Moss, B. 2001. Poxyiridae: the viruses and their replication, p. 2849-2883. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 2. Lippincott/The Williams & Wilkins Co., Philadelphia, Pa.
Non-patent reference 2: Yoshiro Fukuda, Toshitaka Takagi: Pathology of postvaccinal encephalitis, Clinics and Virus 2:53-58, 1974
Non-patent reference 3: So Hashizume: Basis of new attenuated vaccine lymph LC16m8 strain, Clinics and Virus 3:13-19, 1975
Non-patent reference 4: Hashizume S., Yoshizawa H., Morita M., Suzuki K.: Properties of attenuated mutant of vaccinia virus, LC16m8, derived from Lister strain. Vaccinia Virus as Vectors for Vaccine Antigens, Quinnan, Ed. Elsvier Scienced Publishing Col, Inc 87-99, 1985Non-patent reference 5: Hiroko Kuboya: Evaluation for adult inoculation of dried cell culture vaccinia vaccine “LC16•Chiba”, Chiba Prefectural Institute of Public Health Report No. 28, 11-14, 2004Non-patent reference 6: Takahashi-Nishimaki, Funahashi F., Miki S. et al.: Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins. Virology 181: 158-164, 1991Non-patent reference 7: Engelstad M. and Smith G.: The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence. Virology 194, 627-637, 1993Non-patent reference 8: Kidokoro, M., M. Tashiro, and H. Shida. 2005. Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8. Proc. Natl. Acad. Sci. USA 102:4152-4157, 2005Non-patent reference 9: Masayuki Saijo et al., Growth test of LC16m8 strain including LC16mO-type revertant virus in rat skin test, Study subsidy in welfare and labor science, Special Research Project of Welfare and Labor Science, Study on quality management of cell culture vaccinia vaccine (H15-Tokubetsu-43), 2003